Functionalized cyclopropenes as bioorthogonal chemical reporters

Article abstract of DOI:10.1021/ja3060436

Chemical reporters are unique functional groups that can be used to label biomolecules in living systems. Only a handful of broadly applicable reporters have been identified to date, owing to the rigorous demands placed on these functional groups in biolo

Full text of DOI:10.1021/ja3060436

Article
pubs.acs.org/JACS
Functionalized Cyclopropenes As Bioorthogonal Chemical Reporters
David M. Patterson,† Lidia A. Nazarova,† Bryan Xie,‡ David N. Kamber,† and Jennifer A. Prescher*,†,‡,§
‡
§
†Departments of Chemistry, Molecular Biology and Biochemistry, and Pharmaceutical Science, University of California - Irvine,
Irvine, California 92697, United States
S
* Supporting Information
ABSTRACT: Chemical reporters are unique functional groups that
can be used to label biomolecules in living systems. Only a handful of
broadly applicable reporters have been identified to date, owing to the
rigorous demands placed on these functional groups in biological
settings. We describe here a new chemical reportercyclopropene
that can be used to target biomolecules in vitro and in live cells. A variety of substituted cyclopropene scaffolds were synthesized
and found to be stable in aqueous solution and in the presence of biological nucleophiles. Furthermore, some of the
cyclopropene units were metabolically introduced into cell surface glycans and subsequently detected with covalent probes. The
small size and selective reactivity of cyclopropenes will facilitate efforts to tag diverse collections of biomolecules in vivo.
reporter groups. This select class includes ketones, terminal
alkynes, and organic azides.3−6 Azides, in particular, have been
widely utilized in live cells and animals owing to their
remarkable biocompatibility and unique reactivity.2,7,8 Azides
can be readily affixed to metabolic precursors that target
glycans, lipids, and numerous other biomolecules.9−11 Once
installed, these motifs can be selectively reacted with soft
nucleophiles (via Staudinger ligation) or activated alkynes (via
copper-free “click” chemistry) without detriment to the cell or
organism.12−16 Identifying new chemical reporters remains an
important, yet challenging, goal as most functional groups do
not meet the stringent criteria required for use in living systems.
The scaffolds must remain inert to endogenous biological
functionality yet react robustly with complementary probes in
complex environments. Chemical reporters must also traverse
biosynthetic pathways and, thus, be minimally perturbing to the
cell’s metabolic machinery.
INTRODUCTION
■
The bioorthogonal chemical reporter strategy is among the
most popular methods to tag biomolecules in live cells and
whole animals.1 This technique relies on the metabolic
introduction of a unique functional group (i.e., a chemical
reporter) into a biomolecule of interest (Figure 1A). The
In recent years, strained alkenes and alkynes have been
identified that meet several of the criteria for broadly applicable
chemical reporters.8 These scaffolds, including trans-cyclo-
octene (TCO), norbornene (NB), and bicyclononyne (BCN),
are abiotic and relatively stable in cellular environs.17−20
Furthermore, they react rapidly with electron-poor tetrazines
via inverse-electron-demand Diels−Alder (IED-DA) reactions.
The remarkable speed of these reactions is well suited for
sensitive imaging applications, and a variety of TCO- and NB-
conjugated nanoparticles and antibodies have been utilized for
this purpose.21−23 More recently, Chin and others have
demonstrated that amino acids outfitted with BCN, TCO, or
NB can be incorporated into cellular proteins utilizing
engineered strains of bacteria; the functionalized proteins can
be subsequently targeted with visual probes via IED-DA
ligations.21,24,25 While useful, strained alkenes and alkynes have
been slow to transition as reporter groups for other metabolic
Figure 1. Chemical reporters and bioorthogonal chemistries. (A)
Bioorthogonal chemical reporter strategy. A biomolecule of interest
(light-blue rectangle) can be targeted with a chemical reporter group
(red circle) appended to a metabolic precursor (dark-blue rectangle).
Subsequent covalent reaction enables the target biomolecule to be
visualized or retrieved. (B) Cyclopropenes undergo cycloaddition
reactions with tetrazine scaffolds. (C) Panel of cyclopropene analogues
examined in this study.
reporter is detected in a second step using highly selective (i.e.,
bioorthogonal) chemistries.2 Depending on the type of
covalent labeling agent employed, this two-step approach can
be used to visualize biomolecules in cellular environments or
enrich them for further analyses.
While powerful, the bioorthogonal chemical reporter strategy
has been limited to only a handful of broadly functional
Received: June 20, 2012
Published: October 16, 2012
© 2012 American Chemical Society
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pathways. This is due, in part, to their large size and
incompatibility with many endogenous biosynthetic pathways.
We aimed to examine a smaller strained olefincyclo-
propenefor use as a bioorthogonal chemical reporter.
Cyclopropenes are not present in most eukaryotes, and are
likely compatible with a variety of metabolic pathways owing to
their small size. In fact, the steric demand of a cyclopropene
unit is on par with diazirine, a widely used functional group in
cellular labeling and photocross-linking studies.26,27 Cyclo-
propenes also possess a large amount of strain energy that can
drive IED-DA reactions and other cycloadditions under
relatively mild conditions (Figure 1B).28,29 These types of
transformations are particularly attractive for use in biological
settings and have been the subject of recent work by Devaraj
and co-workers.29 In this article, we describe the development
and utilization of cyclopropenes as chemical reporters in living
systems.
Scheme 1. Synthesis of Functionalized Cyclopropenes
RESULTS AND DISCUSSION
■
Design and Synthesis of Biocompatible Cyclopro-
penes. While cyclopropenes possess many favorable attributes
for cell-based studies, they are not without limitation.
Cyclopropene itself is prone to polymerization at room
temperature and susceptible to attack by thiols and other
biological nucleophiles.30,31 However, several lines of evidence
suggest that modifications to the cyclopropene core can
markedly improve scaffold stability. For example, substituted
cyclopropenes are found in both plant and marine natural
products, indicating that C-1- and C-2-modified olefins possess
some degree of metabolic stability.32−34 Methyl-substituted
cyclopropenes are also produced on the ton-scale in the
agricultural industry and used in produce transport.35 Addi-
tionally, carbonyls and other electron-withdrawing groups
positioned at C-3 are known to stabilize cyclopropenes by
imparting partial aromatic character to the ring.36−38
We reasoned that a combination of steric and electronic
modifications to the cyclopropene core would provide a
chemical reporter suitable for metabolic labeling without
compromising cycloaddition reactivity. To test this hypothesis,
we designed a panel of cyclopropenes with vinyl methyl
substituents and various C-3 appendages (Figure 1C). The C-3
groups differed in their electron-withdrawing character and, in
some cases, provided handles for eventual attachment to
metabolic precursors. We were particularly attracted to the
amide- and carbamate-functionalized scaffolds (2 and 11,
respectively) as these linkages mimic those found in numerous
bioconjugates.
Cyclopropenes can be readily accessed from alkynes, but the
synthesis of such low-molecular weight compounds presents
unique challenges. Many cyclopropenes are volatile and, as
mentioned earlier, prone to polymerization upon concen-
tration. To mitigate against these effects, we utilized
disubstituted alkynes in the early stages of our syntheses.
TMS-protected propyne and 2-butyne were first subjected to
rhodium-catalyzed cyclopropenation with ethyl diazoacetate
(5) to provide esters 6a,b (Scheme 1A). Subsequent hydrolysis
of the isolated esters afforded the free acids 1a,b in good yield.
With 1 and 6 in hand, we were poised to access the
remaining C-3-modified scaffolds. The amide-functionalized
cyclopropenes 2a,b were prepared by treating 1a,b with
pentafluorophenyl trifluoroacetate (PFP-TFA), followed by
isopropylamine. The hydroxy-substituted cyclopropenes 3b and
8 were generated via DIBAL-mediated reduction of 6.
Unfortunately, attempts to deprotect 8 to afford the
monosubstituted cyclopropene 3a were unsuccessful. NMR
analyses suggested that 3awith a single methyl substituent
and no electron-withdrawing grouppolymerizes rapidly upon
concentration (data not shown). Last, the carbamate scaffold
11 was isolated in two steps from 3b (Scheme 1B). In contrast
to 3a, cyclopropenes 1, 2, 3b, and 11 exhibited remarkable
stability in aqueous buffer and in the presence of biologically
relevant thiols. Scaffolds 2a and 11, in particular, were found to
be stable for extended periods of time in solution and in the
presence of cysteine (Figures S1, S2 in SI).
Analysis of Cyclopropene−Tetrazine Reactivity. To
examine whether the substituted cyclopropenes were still
amenable to facile cycloaddition, we subjected 1, 2, 3b, and 11
to the model dipyridyl-tetrazine reagent 12. Cycloadduct
formation was observed in all cases when excess cyclopropene
was used (Figure 2A, Figure S3, SI), although the products
formed between 1 and 12 degraded rapidly in solution. The
reactions also exhibited distinct fuchsia-to-yellow color changes
that were used to calculate second-order rate constants for the
transformations (Table 1, Figures S4 and S5 in SI). As
expected, faster reactions were observed in more polar solvents
and with less sterically congested cyclopropenes.29 Additionally,
cyclopropenes with reduced electron-withdrawing character at
C-3 were found to react more expediently with 12, in
agreement with previous studies.36 Scaffolds 2a and 11 also
exhibited comparable reactivity with a functionalized tetrazine
probe.
While the cyclopropene reactions are markedly slower than
other tetrazine-based ligations (∼4−5 orders of magnitude
slower than some TCO reactions),39 they are still suitable for
use in biological systems. In fact, the cycloaddition rates
measured for 2a and 11 are on par with two bioorthogonal
reactions widely used in live cells and animals: the Staudinger
ligation (k = 0.25 × 10−2 M−1 s−1 in 5% H2O/CH3CN) and the
strain-promoted azide−alkyne cycloaddition with a difluori-
nated cyclooctyne (k = 7.6 × 10−2 M−1 s−1 in CH3CN).14,40
These reactions remain popular despite their relatively slow
rates, as the need for small, nonperturbing chemical reporters
(e.g., azides) can often trump the need for rapid reactivity in
living systems.
Our analyses of the cycloaddition reactions also revealed
important mechanistic details. Cyclopropene−tetrazine liga-
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Subsequent ring closure can ultimately alter the position of the
C-3 substituent (placing it over the tetrazine ring, as in 14).
Indeed, when dimethyl cyclopropene 2b was treated with 12,
1
resonances for both 13 and 14 were observed in the H NMR
spectrum. We also noticed that the yellow color of this solution
faded over time in aqueous buffer. NMR and HPLC analyses
revealed that the initial cycloadduct undergoes further intra-
molecular attack at the imine carbon to yield 15 (Figure 2 and
Figures S6−S8 in SI). This internal cyclization was also
observed when cyclopropene 3b was treated with tetrazine 12,
although intramolecular attack proceeded at a faster rate
(Figures S9, S10 in SI). By contrast, when 11 (lacking a suitable
C-3 nucleophile) was treated with 12, no intramolecular
cyclization was observed following ring-opening (Figures S11,
S12 in SI). While some cyclopropene−tetrazine adducts are
prone to further rearrangement, it is important to note that the
starting materials remain covalently linked.
Protein Modification via Cyclopropene−Tetrazine
Ligation. In addition to undergoing rapid and selective
ligation reactions, chemical reporters must function in complex
environments. To evaluate the cyclopropene scaffolds in a
biologically relevant setting, we appended the reporters to a
model protein (lysozyme or BSA). Standard NHS-ester- and
carbonate-coupling reactions were used to attach cyclopropene
scaffolds 10 and 16, respectively, to the protein surface (Figure
3A and Scheme S1 in SI). The modified protein samples were
then reacted with a rhodamine-functionalized tetrazine scaffold
(Tz-Rho, Scheme S2 in SI) and analyzed via mass spectrometry
(Figure S13 in SI) or in gel fluorescence imaging (Figures 3A,
Figure S14 in SI). As depicted in parts B−D of Figure 3 and in
Figure S14 in SI, the ligations were both time- and dose-
dependent, and no reaction was observed in the absence of
either Tz-Rho or cyclopropene.
We also examined whether the cyclopropene−tetrazine
ligation is compatible with azides and strained alkynes. The
orthogonality of such reagents would enable cyclopropenes to
be used in tandem with established bioorthogonal chemistries
for dual labeling experiments. Cyclopropenes are known to
react with organic azides and other 1,3-dipoles, but such
reactions typically require strong heating.44 Indeed, when
cyclopropenes 2a or 11 were subjected to azidoethanol or
phenyl azide in organic solvent, no reaction was observed under
ambient conditions over 24 h (data not shown). Additionally,
Figure 2. Cyclopropenes react with tetrazines to form covalent
adducts. (A) HPLC analysis of the cycloaddition between 2b and 12.
The reaction was initiated in organic solvent prior to the addition of
aqueous buffer. (B) Cyclopropene−tetrazine ligation proceeds via an
initial Diels−Alder reaction, followed by N2 elimination. Subsequent
ring-opening and closing provides a mixture of diastereomers (13 and
14). Intramolecular cyclization ultimately affords the tricyclic adduct
15. Diagnostic NMR chemical shifts are noted.
tions proceed via an initial Diels−Alder cycloaddition, followed
by N2 expulsion. Facial selectively in the initial cycloadduct is
dictated by steric considerations, with the anti-addition product
likely predominating for most cyclopropenes (Figure 2 and
Figures S6−S12 in SI).41 For disubstituted cyclopropenes,
though, the strain associated with multiple suprafacial
substituents can drive further electrocyclic ring-opening.42,43
Table 1. Cycloaddition Rates Observed between Cyclopropene and Tetrazine Scaffolds
entry
cyclopropene
R1
R2
−H
−H
tetrazine
solvent
CH3CN
k (10−2 M−1 s−1
)
1
2
3
4
5
6
7
8
9
2a
2a
2a
2b
3b
3b
11
11
11
−C(O)NH−i-Pr
−C(O)NH−i-Pr
−C(O)NH−i-Pr
−C(O)NH−i-Pr
−CH2OH
12
3.0 0.2
5.1 0.7
1.4 0.2
12
CH3CN/PBS
CH3CN/PBS
CH3CN
−H
Tz-biotin
a
−CH3
−CH3
−CH3
−CH3
−CH3
−CH3
12
ND
12
CH3CN
1.9 0.3
−CH2OH
12
CH3CN/PBS
CH3CN
15
2
−CH2OC(O)NH−i-Pr
−CH2OC(O)NH−i-Pr
−CH2OC(O)NH−i-Pr
12
0.28 0.03
3.5 0.5
3.9 0.5
12
CH3CN/PBS
CH3CN/PBS
Tz-biotin
a
1
Due to slow kinetics, rate was determined by H NMR in CD3OD (k = 0.037 0.006 × 10−2 M−1 s−1).
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observed over 12 h in PBS (Figure S15 in SI). Additionally,
coadministration of Tz-Rho and DBCO-488 to Cp/Az-BSA
did not significantly diminish covalent protein labeling (Figure
3E, Figure S16 in SI). While a detailed analysis of tetrazine-
DBCO reactivity has not been performed, our results suggest
that the two reagents can be used concurrently to target
cyclopropenes and azides under certain conditions.
Metabolic Incorporation of Cyclopropenes onto Live
Cell Surfaces. Beyond biomolecule modification in vitro,
chemical reporters must be able to traverse metabolic pathways
in vivo. This requires that the scaffolds are stable in living
systems and small enough to be tolerated by biosynthetic
enzymes.1 To investigate whether cyclopropenes would be
useful for cellular labeling studies, we constructed a
methylcyclopropene−sialic acid conjugate (9-Cp-NeuAc,
Scheme S1 in SI). Modified sialic acids of this sort are
known to be metabolized by cells and incorporated into cell
surface glycans.47−50 Jurkat cells were incubated with various
concentrations of 9-Cp-NeuAc for 24−48 h. The presence of
cell surface cyclopropenes was subsequently probed by reaction
with a tetrazine−biotin conjugate (Tz-Biotin, Scheme S2 in SI)
and avidin staining (Figure 4A). The fluorescence of each cell
Figure 3. Methylcyclopropenes can be selectively modified on protein
surfaces. (A) Cyclopropenes (Cp) and azides (Az) were appended to
BSA (12.5 mg/mL in PBS) via NHS ester coupling or carbonate
activation (8.4 mM labeling reagent). The labeled proteins (2 mg/mL)
were subsequently reacted with either a tetrazine−rhodamine (Tz-
Rho) conjugate or a cyclooctyne−fluorescein conjugate (DBCO-488).
(B) Gel analysis of cyclopropene-modified BSA incubated with 100
μM Tz-Rho for 0−60 min or no reagent (−). (C) Gel analysis of Cp-
modified BSA labeled with Tz-Rho (0−250 μM) for 1 h. (D) Gel
analysis of Cp-modified BSA (+) or BSA only (−) treated with Tz-
Rho (500 μM) or no reagent (−) at 37 °C for 1 h. (E) Gel analysis of
BSA functionalized with Cp, Az, or both chemical reporters (lanes 10−
13) and reacted for 1 h with either 100 μM Tz-Rho, DBCO-488, both
reagents simultaneously, or no reagent. For B−E, protein loading was
assessed with Coomassie stain (lower panels).
when cyclopropene (Cp)- and azide (Az)-modified BSA
conjugates were mixed together (providing Cp/Az-BSA),
both functional groups could be selectively targeted with
covalent probes (either Tz-Rho or a dibenzocyclooctyne−
fluorophore conjugate, DBCO-488),45 suggesting that Cp and
Az can coexist to a certain extent (lanes 11−13, Figure 3E).
Reduced fluorescence intensities were observed when either
DBCO-488 or Tz-Rho was incubated with Cp/Az-BSA
(compared to BSA samples modified with either Az or Cp
alone), but this was likely due to fewer reporter groups present
in the sample itselfCp-BSA and Az-BSA were combined 1:1
to generate the mixed sample (Cp/Az-BSA), halving the
number of available reporter groups (Figure 3E).
Cyclopropene reactivity with cyclooctynes and other strained
molecules has not been extensively investigated. However,
when cyclopropene-modified BSA (Cp-BSA) was treated with
DBCO-488, no signal above background was observed under
the labeling conditions employed (lane 6, Figure 3E). The faint
fluorescence signal can be attributed to nonspecific DBCO-488
reactivity (lane 3, Figure 3E). By contrast, the compatibility of
tetrazine scaffolds with both azides and strained alkynes has
been examined in more detail.22,24,46 In a recent study,
Hilderbrand and co-workers observed reactivity between a
monosubstituted tetrazine and a DBCO conjugate (k = 6 ×
10−2 M−1 s−1). However, no reaction was observed when a
disubstituted, deactivated tetrazine was employed.22 The
authors further demonstrated that the kinetically slower
tetrazine could be used in tandem with DBCO to label
TCOs and azides in cells. In our studies, Tz-Rho was expected
to react with DBCO (albeit minimally) based on cycloaddition
rates measured for similar tetrazines and TCO.39 However,
when Tz-Rho was incubated with DBCO-488, no reaction was
Figure 4. Cyclopropenes can be metabolically incorporated onto live
cell surfaces. (A) Jurkat cells were incubated with 9-Cp-NeuAc (0−2
mM), a control sugar (NeuAc, 2 mM) or both 9-Cp-NeuAc and
NeuAc (or Ac4ManNAc) for 24 h. After washing, the cells were
reacted with Tz-Biotin (100 μM) for 1 h at 37 °C. Subsequent
staining with APC−avidin and flow cytometry analysis provided the
plots in (B). (C) Mean fluorescence intensities (in arbitrary units, au)
for the histograms in (B). Error bars represent the standard deviation
of the mean for three experiments.
population was measured using flow cytometry. As shown in
Figure 4B, a dose-dependent increase in signal was observed
when cells were incubated with increasing concentrations of 9-
Cp-NeuAc, indicating successful metabolic incorporation of the
chemical reporter. The incorporation efficiency of 9-Cp-NeuAc
was lower than that of a similarly functionalized azido sugar (9-
Az-NeuAc, Scheme S1 in SI) but on par with other unnatural
sialic acids used in metabolic engineering studies (Figure S17 in
SI).47−50 Importantly, the fluorescence signal also diminished
when 9-Cp-NeuAc-treated cells were cultured in the presence
of unlabeled sugars (sialic acid, NeuAc, or peracetylated N-
acetylmannosamine, Ac4ManNAc) targeting the same metabol-
ic pathway.47
We further investigated whether cyclopropene- and azide-
modified sugars could be utilized concurrently for live cell
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Figure 5. Methylcyclopropenes and organic azides can be utilized in tandem for cellular metabolic labeling. (A) Flow cytometry analysis of Jurkat
cells treated with 9-Cp-NeuAc (1 mM), 9-Az-NeuAc (1 mM), or no sugar for 24 h. After washing, a portion of the 9-Cp- and 9-Az-NeuAc cells
were mixed. Cell samples were then washed and subsequently reacted with Tz-biotin (100 μM), DBCO-Rho (100 μM), or both reagents for 1 h at
37 °C. Following staining with APC−avidin, cellular fluorescence was measured. Plots are shown with Rho (azide) and APC (cyclopropene) levels
on the x- and y-axes, respectively. (B) Flow cytometry analysis of Jurkat cells incubated with 9-Cp-NeuAc (1 mM) and 9-Az-NeuAc (1 mM)
simultaneously. After 24 h, the cells were washed, reacted, and analyzed as in (A). For (A) and (B), the same patterns of labeling were apparent in
replicate experiments.
labeling. In one setup, Jurkat cells were incubated with 9-Cp-
NeuAc, 9-Az-NeuAc, or no sugar. After 24 h, portions of the
sugar-treated cells were combined. In a second setup, Jurkat
cells were cultured with both sugars simultaneously. All samples
were subsequently reacted with either Tz-Biotin, a water-
soluble cyclooctyne-fluorophore conjugate (DBCO-Rho), or
both reagents. Cells treated with Tz-Biotin were also stained
with APC−avidin. The fluorescence of the resulting cell
populations was analyzed via two-color flow cytometry, and
the corresponding plots are depicted in Figure 5. For cells
cultured separately with the unnatural sugars prior to mixing
and covalent reaction, flow analysis revealed two distinct cell
populationsone with robust APC fluorescence (correspond-
ing to the 9-Cp-NeuAc-treated cells) and one with robust
rhodamine fluorescence (corresponding to the 9-Az-NeuAc-
treated cells) (Figure 5A). For cells cultured with the
cyclopropenyl and azido sugars simultaneously, treatment
with both covalent probes and flow analysis revealed a single
population of cells labeled with both fluorophores (Figure 5B).
The overall fluorescence signal attributed to the cyclopropene
modification was reduced in this case, although likely due to the
lower incorporation efficiency of 9-Cp-NeuAc compared to
that of 9-Az-NeuAc. Nonspecific reactivity with DBCO-Rho
was also observed in the cell-labeling studies, but importantly,
no cross-reactivity was observed when 9-Az-NeuAc-treated
cells were labeled with Tz-Biotin or when 9-Cp-NeuAc-treated
cells were labeled with DBCO-Rho (Figure S18 in SI).
Collectively, these results suggest that cyclopropene- and azide-
based chemical reporters can be utilized together in live cells
and will be useful for multiplexed metabolic engineering
strategies.
CONCLUSIONS
■
In summary, functionalized cyclopropenes have been developed
for use as chemical reporters in living systems. These scaffolds
react with tetrazines to form covalent adducts in high yield and
with rates suitable for biological labeling applications. Our data
also indicate that cyclopropenes are stable in biological environs
and can be used to derivatize proteins and other biomolecules.
Moreover, these functional groups can be metabolically
introduced into cellular glycans, suggesting they are small
enough to traverse biosynthetic pathways in live cells.
Methylcyclopropenes can also be used in tandem with organic
azides, and we anticipate that combinations of these and other
chemical reporters will be widely used for targeting multiple
classes of biomolecules.51
This work also sets the stage for continued expansion of the
bioorthogonal chemistry toolkit. We envision developing a
collection of cyclopropene scaffolds suitable for use as both
chemical reporters and secondary labeling agents. Toward this
end, we are generating cyclopropenes that react more rapidly
with tetrazine probes, along with identifying scaffolds with
alternative modes of reactivity. These reagents will bolster
efforts to monitor multicomponent biomolecular processes in
living systems.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details, full spectroscopic data for all new
compounds, and additional images. This material is available
free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
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jpresche@uci.edu
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
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This work was supported by the UC Irvine School of Physical
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We also thank Drs. John Greaves and Beniam Berhane for
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